PROJECT SUMMARY/ABSTRACT Goals: Questions regarding the role of chromosome organization on inheritance and gene expres- sion raise many technical challenges. Not only is chromosomal DNA the largest biomolecule in the cell, it is comprised of segments that fold independently in ways that vary from cell to cell. Furthermore, ex- cepting the X and Y, chromosomes of diploid organisms come in pairs of homologs that defy easy dis- tinction via imaging. This application proposes a suite of tools for tracing the path of entire chromo- somes at the single cell level in a sequence- and homolog-specific fashion using widefield, confocal, as well as super-resolution microscopy. As one aspect of the tools are libraries of oligonucleotides (oligos) which, while renewable, are expensive, such libraries will be designed to be broadly useful so that they can be shared freely among laboratories. In brief, this proposal aims to provide new tools that will ad- vance our understanding of the relationship between chromosome function and chromosome organiza- tion. Health relatedness: The organization of the genome within the nucleus and the progression of that organization through development has profound consequences for gene expression and, thus, development and disease; chromosome topology and interchromosomal interactions affect enhancer- promoter interactions, global silencing, chromosome stability and repair, chromosome segregation, and, thus, even inheritance. As such, single-cell imaging-based studies that elucidate how the genome is organized will do much to further our understanding and treatment of disease. Innovation: The proposed aims are focused heavily on the development of new technologies and, thus, they hold promise for enabling studies and discoveries that might not otherwise be possible. In particular, they aim to facilitate the imaging of chromosomes in situ by improving probe effectiveness and probe design, increasing speed of data acquisition and quantity of data collected, and enhancing image resolution. In this way, they may provide the community with tools that can render complex genomes, such as those of humans, more accessible for analysis. In brief, the aims are dedicated to: Aim 1 Optimizing Oligopaints Aim 2 Tracing chromosomes using Oligopaints, OligoSTORM, and OligoDNA-PAINT Aim 3 Tracing chromosomes using OligoFISSEQ